The proposed research described in this application deals with three mitochondrial enzymes whose deficiency leads to clinically significant inherited metabolic disease in man - methylmalonyl-CoA mutase (MUT), propionyl-CoA carboxylase (PCC), and ornithine transcarbamylase (OTC) - and with Bcl-2, a putative mitochondrial protein with roles in apoptosis and oncogenesis. The experiments described are designed: 1) to identify, at the molecular level, mutations in MUT, PCC, and OTC which lead to metabolic disease and to express the variant proteins in order to determine the effects of the mutations on the biochemistry and biogenesis of these enzymes; 2) to compare this data with that from the three-dimensional structure of MUT and OTC to establish structure/function relationships, to test in a biological system any hypotheses based on this data, and to correlate the mutations and their effects with patient phenotypes; and 3) to evaluate further the proposed mitochondrial localization of Bcl-2, including the characteristics of its interaction, its submitochondrial location, and the effects of its overexpression on mitochondrial transport and metabolism. The methods used to approach these problems will include polymerase chain reaction (PCR) amplification of cDNA and genomic DNA from fibroblast cell lines from patients, direct DNA sequencing of the PCR products, and expression of the mutant proteins in E. coli, S. cerevisiae, or HeLa cells. Biochemical analysis of enzyme kinetics and stability will define the effects of some mutations, while cell biological experiments will delineate their impact on enzyme translocation, folding, and assembly. The characteristics of the association of Bcl-2 with mitochondria will be explored by in vitro translocation studies with isolated mitochondria and mitochondrial sub-fractions, and the impact of Bcl-2 overexpression on mitochondrial metabolism and transport will be evaluated in intact cultured cells. These investigations are expected to provide new information on the function of individual amino acids in the mechanism of enzyme reactions, on the pathogenic effects of mutations on physiology and homeostasis, and on the role of mitochondria in mediating apoptosis and oncogenesis.